The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-β peptide fibrils and inhibits secondary nucleation

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD β-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-β (Aβ42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aβ, the CTD constructs instead bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two β-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization

[Image: see text] α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation

Capturing transient peptide assemblies associated with Alzheimer's disease : Native mass spectrometry studies of amyloid-β oligomerization

Correct folding of proteins is essential for maintaining a functional living cell. Misfolding and aggregation of proteins, where non-native intermolecular interactions form large and highly ordered amyloid aggregates with low free energy, is hence associated with multiple diseases. One example is Alzheimer’s disease (AD) where the Amyloid-β (Aβ) peptide aggregates into amyloid fibrils, which deposit as neuritic plaques in the brains of AD patients. Nucleation of amyloid fibrils takes place via formation of smaller pre-nucleation clusters, so-called oligomers, which are considered to be especially toxic and are therefore potentially important in AD pathology. Detailed mechanistic molecular knowledge of Aβ aggregation is important for design of AD treatments that target these processes. The oligomeric species are however challenging to study experimentally due to their low abundance and high polydispersity.   Aβ oligomers are in this thesis studied under controlled in vitro conditions using bottom-up biophysics. Highly pure recombinant Aβ peptides are studied, primarily using native ion-mobility mass spectrometry, to monitor the spontaneous formation of oligomers in aqueous solution. Mass spectrometry is capable of resolving individual oligomeric states, while ion mobility provides low-resolution structure information. This is complemented with other biophysical techniques, as well as theoretical modeling. The oligomers are also studied upon modulating intrinsic factors, such as peptide length and sequence, or extrinsic factors, such as the chemical environment. Interactions with two important biological interaction partners are studied: chaperone proteins and cell membranes.   We show how Aβ oligomers assemble, and form extended structures which may be linked to continued growth into amyloid fibrils. We also show how different amyloid chaperone proteins interact with growing aggregates, which modifies and delays the aggregation process. These interactions are shown to depend on specific sequence-motifs in the chaperones and client peptides. Membrane-mimicking micelles are on the other hand able to stabilize globular compact forms of the Aβ oligomers and to inhibit the formation of extended structures which nucleate into amyloid fibrils. This may contribute to enrichment of toxic species in vivo. Interactions with membrane-mimicking systems are shown to be highly dependent on both the Aβ peptide isoform and the properties of the membrane environment, such as headgroup charges. It is also demonstrated how addition of a designed small peptide construct can inhibit formation of Aβ oligomers in the membrane environment.

Разработка способов модернизации непрерывной раскатки бесшовных труб с целью повышения качества трубного проката

Отчет о НИР (заключительный). Руководитель темы Ю. Л. Бобарикин

Evacuation for an automatic transportation system in Sweden

En utrymningsplan skall utformas till ett befintligt automatiskt persontransportsystem. Lösningen skall vara såväl attraktiv som säker. Det automatiska transportsystemet kan befinna sig i olika geografiska nivåer, vilket gör att en generell utrymningslösning över hela systemet blir mycket svår att ta fram. Detta för att fordonet kan befinna sig fem meter ovanför mark, i marknivå eller under marknivån. Syftet med denna rapport är att fastställa vilka säkerhetsrisker som kan uppkomma i ett automatiskt transportsystem, hur systemet och resenärer i systemet bör agera vid dessa händelser samt presentera idéer på hur resenärerna ska kunna evakueras från vagnen och systemet då det är nödvändigt att utrymma. I detta projekt så har inget större hänseende tagits till den ekonomiska kalkylen och inte heller till hur implementering av lösningarna ska realiseras. En scenarioanalys och probleminventering har genomförts för att fastställa de olika scenarion som kan uppkomma och vad följderna hypotetiskt skulle bli ifall respektive händelse inträffade. För att se över riskerna vid ett scenarie så har en riskanalys genomförts.An evacuation plan is to be designed to an existing automatic transportation system for personal transport. The solution should be attractive as well as safe and secure. The automatic transportation system is located at different geographical levels. Therefore, a general solution would be very difficult to construct for the whole system, when the vehicles can be located fifteen feet, or more, above the ground, at ground level or below ground level. The purpose of this report is to identify safety risks that can be originated from the automatic system, how the system and the passengers in the system should respond to these events and to present ideas how passengers should be evacuated from the vehicle and the system when it is necessary to evacuate. In this project, no greater regard for the economic calculations is taken into account, nor how the implementation process of the presented solutions will be realized. A scenario analysis and record of issues has been conducted to determine the different scenarios that may arise and what the consequences would hypothetically be if it occurred. A risk analysis has been conducted to evaluate the risks of an event

Minimerandet av UCL skador för professionella skidåkare

This report is a Master thesis in product development taking on the problem called a "skier´s thumb". A number of different strategies have been proposed to address this issue over the years, however, without much success. One of the most common injuries for professional skiers or ski enthusiast is damaging the Ulnar Collateral Ligament (UCL), or injuring one of the thumbs interphalangeal joint. These types of injuries are usually undetected: moreover, if not treated by professionals they could cause chronic damages to the thumb. To investigate the issue, extensive research was conducted to understand the medical issues that arise when injuries occur. Our goal is to create a solution where the thumb does not move more than 35° angulation, or has a difference of 15° from contralateral thumb. Presented in the report is a selection of results from research and interviews. Additionally, through various ideation and rapid prototyping techniques different ideas were tested to create a feasible product that minimizes injuries on the thumb. Later on, testing of all prototypes was performed to provide a functional concept that could decrease injuries of the thumb. The result of this project is a UCL protecting gear, integrated inside the glove.Denna rapport är ett examensarbete inom produktutveckling som tar sig an problemet "Skidåkartumme". Flera olika strategier har genom åren föreslagits för att lösa denna skada på tummen, utan någon större framgång. En av de främsta skadorna som drabbar professionella skidåkare eller skid-entusiaster, är skador på det ulnara kollateral ligamentet (UCL) eller på någon av tummens knogleder. Dessa skador behandlas oftast inte då skidåkare inte märker av att ligamentet har fått en mindre ruptur. En allvarlig olycka mot UCL kan ge kroniska skador i tummen som leder till viss invaliditet. För att undersöka problemet har omfattande forskning genomförts för att förstå det medicinska problem som uppstår samt för att klargöra rörelseschemat vid en olycka. Projektets mål var att frambringa ett koncept som förhindrar tummens rörelseomfång i skadliga riktningar, 35 graders vinkling eller 15 graders vinkling av tummens falang. Rapporten presenterar resultat från forskning samt intervjuer och även framtagning av koncept genom olika idégenereringar och avancerade prototyp-tillverkningstekniker. Vidare testades samtliga prototyper för att få fram ett funktionellt koncept som kan förebygga skador mot tummens ligamanet. Resultatet av detta arbete är en funktionell prototyp, där skyddet som förhindrar skador mot UCL är integrerat i skidhandsken

Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties

Cell-penetrating peptides (CPPs) with sequences derived originally from a prion protein (PrP) have been shown to exhibit both anti-prion and anti-amyloid properties particularly against prion proteins and the amyloid-β (Aβ) peptide active in Alzheimer’s disease. These disease-modifying properties are so far observed in cell cultures and in vitro. The CPP sequences are composed of a hydrophobic signal sequence followed by a highly positively charged hexapeptide segment. The original signal sequence of the prion protein can be changed to the signal sequence of the NCAM1 protein without losing the anti-prion activity. Although the detailed molecular mechanisms of these CPP peptides are not fully understood, they do form amyloid aggregates by themselves, and molecular interactions between the CPPs and PrP/Aβ can be observed in vitro using various spectroscopic techniques. These initial intermolecular interactions appear to re-direct the aggregation pathways for prion/amyloid formation to less cell-toxic molecular structures (i.e., co-aggregates), which likely is why the disease-inducing PrP/Aβ aggregation is counteracted in vivo

Amyloid-β oligomers are captured by the DNAJB6 chaperone : Direct detection of interactions that can prevent primary nucleation

A human molecular chaperone protein, DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), efficiently inhibits amyloid aggregation. This inhibition depends on a unique motif with conserved serine and threonine (S/T) residues that have a high capacity for hydrogen bonding. Global analysis of kinetics data has previously shown that DNAJB6 especially inhibits the primary nucleation pathways. These observations indicated that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-β symbol (Aβ) peptide but with aggregated species. However, these pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here, we employed a native MS-based approach to directly detect oligomeric forms of Aβ formed in solution. We found that WT DNAJB6 considerably reduces the signals from the various forms of Aβ (1-40) oligomers, whereas a mutational DNAJB6 variant in which the S/T residues have been substituted with alanines does not. We also detected signals that appeared to represent DNAJB6 dimers and trimers to which varying amounts of Aβ are bound. These data provide direct experimental evidence that it is the oligomeric forms of Aβ that are captured by DNAJB6 in a manner which depends on the S/T residues. We conclude that, in agreement with the previously observed decrease in primary nucleation rate, strong binding of Aβ oligomers to DNAJB6 inhibits the formation of amyloid nuclei

A Hairpin Motif in the Amyloid-β Peptide Is Important for Formation of Disease-Related Oligomers

The amyloid-β (Aβ) peptide is associated with the development of Alzheimer’s disease and is known to form highly neurotoxic prefibrillar oligomeric aggregates, which are difficult to study due to their transient, low-abundance, and heterogeneous nature. To obtain high-resolution information about oligomer structure and dynamics as well as relative populations of assembly states, we here employ a combination of native ion mobility mass spectrometry and molecular dynamics simulations. We find that the formation of Aβ oligomers is dependent on the presence of a specific β-hairpin motif in the peptide sequence. Oligomers initially grow spherically but start to form extended linear aggregates at oligomeric states larger than those of the tetramer. The population of the extended oligomers could be notably increased by introducing an intramolecular disulfide bond, which prearranges the peptide in the hairpin conformation, thereby promoting oligomeric structures but preventing conversion into mature fibrils. Conversely, truncating one of the β-strand-forming segments of Aβ decreased the hairpin propensity of the peptide and thus decreased the oligomer population, removed the formation of extended oligomers entirely, and decreased the aggregation propensity of the peptide. We thus propose that the observed extended oligomer state is related to the formation of an antiparallel sheet state, which then nucleates into the amyloid state. These studies provide increased mechanistic understanding of the earliest steps in Aβ aggregation and suggest that inhibition of Aβ folding into the hairpin conformation could be a viable strategy for reducing the amount of toxic oligomers